This invention relates in general to separation systems and, in particular, to a system for measuring and/or controlling electroosmosis in separation techniques.
Capillary separation techniques such as capillary zone electrophoresis (CZE) involving columns filled with liquid have become important analytical techniques for the analysis of various complex sample mixtures. When an electrolyte is introduced into a capillary column electrokinetic separation techniques, the interaction of the electrolyte with the walls of the capillary column may cause the wall to acquire a net charge. When an electrical potential is applied across the two ends of the capillary column holding the electrolyte therein, this net charge will cause the electrolyte to move under the influence of the electric field in a process known as electroosmosis. In addition, different components of the sample have different electrophoretic migration mobility under the influence of the electric field caused by the application of electrical potential across the tube. Therefore, even though the sample may include oppositely charged components migrating electrophoretically in opposite directions, if the electroosmotic flow rate is greater than the migration rate of the sample components migrating in the opposite direction to the above flow, which is usually the case, all the sample components will move in the same direction and separate into zones. Electroosmosis is employed in electrokinetic separation techniques, such as CZE and micellar electrokinetic chromatography.
The time of analysis of samples and the resolution of separation between sample components in CZE depends on the magnitude and direction of electroosmotic flow. For example, it has been shown theoretically that the best resolution between two compounds or sample components is obtained when the electroosmotic flow just counterbalances the average electrophoretic mobility of the two analytes. When this condition is met, it is possible to separate compounds with extremely small differences in mobility. Therefore, the ability to control and manipulate the electroosmotic flow may be beneficial for the separation and analysis of samples in CZE and micellar electrokinetic chromatography.
Several techniques have been developed for altering the electroosmotic flow. Such methods involve using coated or surface-modified capillaries or adding special compounds to the separation buffer. Still other methods employ coated and uncoated capillaries placed in tandem; see Nashabeh et al., J. High Res. Chromatogr., Vol. 15, page 289 (1992). Yet other methods involve the application of external electric fields; see Lee et al., Anal. Chem., Vol. 62, page 1550 (1990), Lee et al., Anal. Chem., Vol. 63, page 1519 (1991), and Hayes et al., Anal. Chem., Vol. 64, page 489A (1992).
None of the above-described methods for altering the electroosmotic flow rate is entirely satisfactory. The use of coated capillaries limits one to separation electrolytes that are compatible with the coating on the capillary surface and hence restricts the range of control of electroosmosis. For example, pH extremes cannot be employed for most coatings. Buffer additives may interact with the analytes of interest to hinder separation and to broaden peaks. This, for example, happens when tetradecyltrimethyl-ammonium bromide (TTAB) is added to negatively charged analytes. The use of multiple capillaries in tandem requires the alignment of capillary inner-channels, and also requires alterations of column lengths each time a change in the electroosmotic flow is desired. The use of external electric fields increases the complexity of instrumentation and has been shown not to work effectively under certain conditions, such as after the column has been rinsed with KOH solution. It is therefore desirable to provide an improved technique for controlling the electroosmotic flow and altering the flow rate in electrokinetic separation.
Also, when certain samples are analyzed, sample components may cause changes in the electroosmotic flow rate. For such samples, in order for the separation to be reproducible with the same results, it is important to to be able to monitor the electroosmotic flow rate in real time. It is therefore desirable to provide a design capable of monitoring the electroosmotic flow rate in real time.